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REVIEW OF LITERATURE
Due to rapid deforestation and depletion of genetic stocks, concerted efforts must be made to
evolve new methods for mass propagation and production of plant species which are high
yielding, resistant to pest and disease associated with increased photosynthetic efficiency.
Conventional breeding is rather slow and less productive and cannot be used efficiently for
the mass multiplication and genetic improvement of trees. To circumvent this, plant tissue
culture and genetic transformation methods offer an important option for effective
multiplication and improvement of plant species. The technology of plant tissue culture offers
advantages over conventional methods of propagation for a rapid and large scale
multiplication of important plants under in vitro conditions irrespective of season with
conservation of space and time (Nehra and Kartha, 1994; Rao et al., 1997). This technique
provides rapid multiplication of selected superior varieties. During the past decade, major
advances have been made in this field and now it has become an industrial technology. An
overview of work carried out earlier on the different plant species have been given in the
following sections.
2.1 Micropropagation
In nature, the methods of plant propagation may be either asexual or sexual. Sexually
propagated plants demonstrate a high amount of heterogeneity since their seed progeny are
not true-to-type unless they have been derived from inbred lines. Asexual reproduction, on
the other hand, gives rise to plants which are genetically identical to the parent plant and thus
permits perpetuation of the unique characters of the cultivars. Multiplication of genetically
identical copies of a cultivar by asexual reproduction is called clonal propagation. When
clonal propagation is through tissue culture, it is popularly called micropropagation.
Advanced biotechnological methods of culturing plant cells and tissues should provide new
means for conserving and rapidly propagating valuable, rare and endangered forest tree
species. The application of micropropagation techniques as an alternative mean of asexual
propagation of important plant species has increased the interest of workers in various fields.
There is also a conservation use for those species that are at risk, rare, endangered or of
special cultural, economic or ecological value (Benson, 2003). The potential benefits of
micropropagation of elite genotypes for production of clonal planting stock for
8
afforestation/reforestation have long been recognized (Winton, 1971; Whitehead and Giles,
1977). In general woody trees are difficult to regenerate under in vitro conditions.
The difficult-to-root recalcitrant species for which there is a serious dearth of planting
material are multiplied through tissue culture and made available for the afforestation
programme. Plant Tissue Culture is a technique by which any plant part can be cultured on a
nutrient medium under sterile conditions with the purpose of obtaining growth. In vitro
methods can be used to produce, maintain, multiply and transport pathogen free plants safely
and economically. This technology is being extensively used for large-scale production of
elite planting material of desired characteristics.
The pioneering experiments were initiated by the father of tissue culture
Gottlieb Haberlandt in 1898, chose single cell, isolated from the palisade tissue of
leaves, epidermis and epidermal hairs of different plants. He grew them on Knop's
(1865) salt solution with sucrose and observed obvious growth in the palisade cells
but could not succeed because of handling with highly differentiated cells and lack
of proper techniques. Hanning (1904) successfully cultured embryos of Raphanus
sativus, Raphanus landra, Raphanus caudatus and Cochlearia danica on Tollen's
medium and obtained transplantable seedlings. The first commercial use of plant
tissue culture on artificial media was in the germination and growth of orchid
plants, in the 1920’s. White (1934) was the first to achieve success in organ cultures of
tomato root tips. The first calli from cambial explants of angiosperms and conifers
trees was observed by Gautheret (1934). Gautheret, White and Nobecourt (1939) laid the
foundation for further work in the field of plant tissue culture. The tissue culture media,
presently in use were modifications of those established by these three pioneers. Ball (1946)
developed the first whole plants through tissue culture from shoot tips of Lupinus and
Tropaeolum. There was a great deal of research in 1950-60, but it was only after the
development of a reliable artificial medium (Murashige & Skoog, 1962) that plant
tissue culture really ‘took off’ commercially. Morel (1950) successfully cultured
monocot plant tissue with the help of coconut milk. Miller et al., (1955) discovered
and isolated 6-furfuryl amino purine and named it as Kinetin. Torrey (1957) and Muir et
al., (1958) demonstrated the single cell proliferation into callus. The shoot and root
initiation in cultured callus can be regulated by varying ratio of auxins and
cytokinins in the medium (Skoog and Miller, 1957). The first successful report on
the formation of somatic embryos from carrot tissue was achieved by Steward
(1958) and Reniert (1959). Some other techniques which controls plant
9
regeneration and morphogenesis in culture included: production of virus free plants
(Morel, 1960), haploid plant production (Guha and Maheswari, 1964), production
of secondary metabolites (Kaul and Staba, 1967), protoplasts fusion by
polyethylene glycol (Kao et al., 1974). The first complete plants from tissue
culture of tree species were regenerated by Winton (1968) from leaf explant of
Populus trichocarpa. Micropropagation by the method of multiple shoot production
directly from seeds has been reported in many herbaceous and woody species (Rodriguez,
1982).
Plant tissue culture is an important alternative to conventional method of vegetative
propagation mainly for raising of elite and rare species (Rao et al., 1996). Plant tissue culture
provides a best tool for large scale production of propagules especially in case of endangered
medicinally important plant species where explant material is available in a very small
quantity. Micropropagation of mature trees employing vegetative explants has been a difficult
task and lagging behind that of herbaceous plant due to various factors, like juvenility,
maturity, inherent slow growing habit, exogenous and endogenous infection, presence of
phenolic compounds, long complex life cycles and great genetic variations (Bonga and
Durzan, 1986; Durzan, 1985; Zimmermen, 1985; Bajaj, 1991). During the last few years,
micropropagation techniques have been used for the rapid and large scale propagation of a
number of fruit and forest trees (Hutchinson and Zimmerman, 1987). Several woody species
such as poplars, wild cherry, eucalyptus, red wood, radiate pine and teak are at present
commercially micropropagated (Thorpe, 1990; Bajaj, 1997). In vitro propagation of forest
tree species is an effective way to capture genetic gain and produce large amounts of plant
material.
It is well established that in vitro propagation of plant species is influenced by several
factors, like genotype, age and source of initial tissue/organ which in turn are related to their
endogenous hormonal status (George, 1993). Tissue culture techniques are also used for
virus eradication, genetic manipulation, somatic hybridization and other procedures
that benefit propagation, plant improvement and basic research. A large number of
horticultural plantations, forest species, important fruit trees and medicinal plant are being
propagated in vitro on commercial scale (Bhojwani and Arumugam, 1993). There are number
of factors that affect the success of in vitro regeneration are discussed here.
10
2.1.1 Selection of explants
The manipulability of organ formation in tissue culture is often determined by the choice of
the explants. While it is generally accepted that totipotentiality is characteristic of all plant
cells. Several plant parts can be used as explants to initiate a plant tissue culture. However,
each plant organ differs in its rate of growth and regeneration because the cells in that organ
exist in a particular developmental stage. Organs also differ in their metabolic activity and
capacity to transport and utilize growth regulators. Generally, meristematic tissues such as the
root and stem tips and auxiliary buds are good explants because they show the most rapid rate
of cell division. Also, these tissues have a greater ability for the uptake and concentration of
growth regulators. The regeneration potential of explants is attributed by the physiological
state, age and cellular differentiation among the constituent cells (Murashige, 1974).
The influence of plant material on the growth and development in tissue culture are related to
many factors such as genotype, age of the plant, age of the tissue or organ, physiological state
of the explants, the state of health of the plant, effect of season throughout the year such as
winter and summer, growth condition such as photoperiod, position of explant within the
plant, size of the explants, wound surface area, method of inoculation, etc. The use of young
and meristematic tissue as in many cases enabled raising of regenerative cultures when
mature and differentiated explants failed to give such response. Explants cells already in an
active process of division contribute to increase in the adventitious bud induction on the
explants (Mohammed et al., 1992: Thome et al., 1995). Juvenility is one of the most
important factor influencing the in vitro response of many woody species (Bonga, 1987).
Nodal explants from mature plants have been used for regeneration in many
tree species like as: Prosopis juliflora (Nandwani and Ramawat, 1991), Dalbergia sissoo
(Gulati and Jaiwal, 1996), Ficus religiosa (Deshpande et al., 1998), Sterculia urens
(Sunnichan et al., 1998), Madhuca latifolia (Bansal and Chibbar, 2000), Melia azedarach
(Shahzad and Siddique, 2001), Toona ciliata (Mroginski et al., 2003), Belanites
aegyptica (Ndoye et al., 2003), Michelia champaca (Iyer et al., 2005), Cassia
angustifolia (Siddique and Anis, 2007), Searsia dentata (Prakash and Staden, 2008), Morus
alba (Balakrishnan et al., 2009), Balanites aegyptiaca (Siddique and Anis, 2009), Arbutus
unedo (Gomes and Canhoto, 2009), Azadirachta indica (Arora et al., 2010), Spondias
mangifera (Tripathi and Kumari, 2010), Acacia auriculiformis (Girijashanker, 2011)
Dalbergia sissoo (Ali et al., 2012).
11
Mehra and Cheema (1980) reported multiple shoots induction from nodal segments
of Populus. Sharma et al., (1984) established callus cultures on modified MS
medium from axillary buds and shoot tips of Phoenix dactylifera. Rai and
Jagdishchandra (1987) induced multiple shoots directly from seeds and seedling
explants of Cinnamomum zeylanicum. Different explants viz. stem, root, leaf and
hypocotyl segments and axillary buds were used for regeneration in Albizzia
lebbeck (Arya et al., 1978; Bhargava et al., 1981). Sharma et al., (1988) reported
the somatic embryogenesis and plant regeneration from shoot tip derived callus of
Phoenix sylvestris. Gharyal and Maheshwari (1990) observed callus formation from
stem and petiole explants of Cassia fistula and Cassia siama and its differentiation
in to shoot buds. Nandwani and Ramawat (1991) reported plantlets regeneration
from nodal segments of Prosopis juliflora.
Rout and Das (1993) reported multiple shoots induction from apical and
axillary meristem explants derived from seedlings of Madhuca longifolia. Perez-
Parron et al., (1994) used shoot tips and nodal segments of Fraxinus angustifolia
for direct regeneration. Mohan et al., (1995) achieved regeneration from hypocotyl
and cotyledonary node explants of Moringa pterygosperma. Purohit and Dave
(1996) reported multiplication of Sterculia urens through cotyledonary node
segments. Micropropagation protocols for Cinnamomum camphora were developed by
Babu et al., (1997) and Huang et al., (1998). Ajithkumar and Seeni (1998) observed direct
organogenesis from nodal, leaf, shoot tip and internode segments of Aegle marmelos. The
protocol for micropropagation of Lagerstroemia reginae using shoot bud culture was
developed by Sumana and Kaveriappa (2000). Sheeja et al., (2000) reported
micropropagation of Cinnamomum verum from mature nodal segments. Indirect
organogenesis from nodal explants of Melia azedarach was reported by Shahzad and
Siddiqui (2001). Multiple shoots were initiated from cotyledonary node segments of Acacia
catechu (Sahni and Gupta, 2002). Romano et al., (2002) has developed an in vitro
propagation protocol based on axillary bud proliferation for mature female trees of Ceratonia
siliqua in MS medium supplemented with BAP or Zeatin. Martin (2003) developed a
protocol for in vitro regeneration of rare woody aromatic medicinal plant-Rotula
aquatica by means of direct and indirect organogenesis. Shou et al., (2005) developed
a protocol for micropropagation of Cinnamomum camphora by the formation of adventitious
shoots from the delicate caudex. Iyer et al., (2005) reported multiple shoots from nodal
12
explants of Michelia champaca. Pandey et al., (2006) developed a protocol for the
regeneration of complete plantlets from nodal explants of Terminalia arjuna.
Pasqual and Ferreira (2007) developed pathogen free plantlets of Ficus carica
through micropropagation. Multiple shoots were produced from mature nodal
segments of Syzigium cumini (Remashree et al., 2007). Soulange et al., (2007)
reported multiple shoot induction in Cinnamomum camphora. Sujatha and Hazra, (2007)
used MS basal medium for induction of multiple shoots from mature-tree-derived
axillary meristems of Pongamia pinnata. Khalafalla and Daffalla (2008) reported
multiple shoots from cotyledonary node segments of Acacia senegal.
Micropropagation protocol has been developed by different workers for efficient plantlet
regeneration through nodal segments of different plants viz. Eucalyptus polybractea
(Goodger et al., 2008) Phoenix dactylifera (Aslam and Khan, 2009), Melia azedarach
(Hussain and Anis, 2009), Arbutus unedo (Gomes and Canhoto (2009), Oroxylum indicum
(Gokhale and Bansal, 2009). Mostafa et al., (2010) developed a protocol for
micropropagation of Arbutus andrachne using explants from seedlings. Similarly, Tripathi
and Kumari (2010) developed an efficient in vitro regeneration protocol for Spondias
mangifera using cotyledonary nodes. Micropropagation from nodal explants from epicormic
shoots of Dalbergia sissoo was developed by Thirunavoukkarasu et al., (2010). Tyagi et al.,
(2010) reported the in vitro regeneration of Crataeva adansonii through nodal segments.
Regeneration of Gomortega keule through zygotic embryos was reported by Mun˜oz-Concha
and Davey (2011). Khalafalla et al., (2011) also reported the intact embryo cultures of
Boscia senegalensis. Clonal propagation of Acacia auriculiformis through axillary
buds was reported by Girijashankar (2011). An efficient and improved in vitro
propagation method for Terminalia catappa has been developed from nodal explants
(Phulwaria et al., 2012). Mehta et al., (2012) developed a protocol for rapid
micropropagation and callus induction of Terminalia bellerica.
Seasonal changes greatly influence explants establishment (Siril and Dhar, 1997).
Seasonal conditions at the time of explants collection may influence the in vitro growth of
explants, phenolics exudation and degree of contamination. Most vegetative propagation
techniques relaying on morphogenetic process are conditioned by the season (Hartman and
Kester, 1986). There is increasing evidence that seasonal differences influence the regulation
of cell cycle and this can affect morphogenetic processes (Anderson et al., 2001). Gupta et
al., (1980) noticed the seasonal effects on regeneration of Tactona grandis. The nodal
13
segments of Eucalyptus tereticornis collected during July to September were more responsive
because of negligible phenolic exudation from explants as compared to that collected in
October-November and May-June due to high amount of phenolic exudation (Das and Mitra,
1990). Excellent regeneration has been reported in plant species during spring season
(March-May) when reserve food material (carbohydrate) is made available and helps plants
sprout and bloom (Bhatt and Todaria, 1990). The nodal explants harvested during the months
of March-April and August-October was found to be the best for cultures establishment of
Capparis decidua (Deora and Shekhawat, 1995). Bansal and Chibbar (2000) observed best
response from nodal segments of Madhuca latifolia in the month of May. The collection of
explants during a relatively milder weather condition (December to March) was best for
promoting survival of explants in Acacia sinuata (Vengadesan et al., 2003). Sharma et al.,
(2003) also observed best explants response in the month of October and November with
maximum number of shoot buds in Crataeva adansonii. The best shoot initiation response
was reported from November to February when the trees produced fresh sprouts; the shoot
initiation was rare in explants collected during other periods in Callophyllum apetalum (Nair
and Seeni, 2003). Nodal explants of Myrica esculenta collected during winter (November-
December) gave the maximum response (Bhatt and Dhar, 2004). The nodal segments of
Wrightia tinctoria collected during March-June from young lateral branches showed
maximum bud break response (Purohit and Kukda, 2004). In Holarrhena antidysenterica,
nodal explants showed maximal morphogenic response from May to July, and declined in
subsequent months till dropping to zero from October to February (Kumar et al., 2005).
Singh and Goyal (2007) observed that the season between August-October was best for
explant collection in Salvadora oleoides. Pati et al., (2008) observed that nodal explants of
Aegle marmelos excised during September-October was found ideal because most of the
explants shows bud break whereas, bud break frequency reduced in other months. The
cultures of Melia azedarach initiated during March exhibited the best response not
only in terms of the frequency of bud break but also in shoot vigor (Husain and Anis,
2009). The explants of Maerua oblongifolia collected during the months of July-August
responded best in vitro as compared to explants harvested in any other months of the year
(Rathore and Shekhawat, 2011). The nodal, inter-nodal segments and shoot apices of Ficus
religiosa collected in May and June gave maximum response (Siwach et al., 2011).
The variation in regenerative behaviour among explants is sometimes
attributable to the age of the tissue or organ and the extent to which constituent
14
cells are differentiated. Thorpe and Biondi (1984) also reported that the
physiological state of the tree has been known to influence the behaviour of
explant in culture. The capacity for clonal propagation is closely linked with the
genetic and physiological factors that control the transition from juvenile to mature
growth in trees (Bonga, 1982). In general, juvenile tissue will better respond to in
vitro treatments.
Explants from mature trees of Eucalyptus citriodora required pre-treatment
for induction of shoot buds but explants from seedlings did not require any pre-
treatment (Gupta et al., 1981). Gulati and Jaiwal (1996) reported that nodal
explants from coppied shoots of mature Dalbergia sissoo exhibited least phenolic
exudation and responded better shoot regeneration than the explants obtained from
mature tree. Philomina and Rao (1999) reported multiple shoots from seed culture
of Sapindus mukorossi. Jha et al., (2002) observed the adventitious shoots formation from
the cotyledonary node explants of Sesbania rostrata. Axillary buds of Crataeva
adansonii taken from the root stock growths proliferated better in comparison to
buds taken from the mature tree (Sharma et al., 2003). Chowdhary et al., (2004)
reported that the nodal explants of Dendrocalamus strictus taken from the 1st and
3rd
positions from base of the secondary branches showed better response
compared to the 4th to 6th positions. This was due to differences in the
physiological states of the two explants. Shoot cultures of Toona ciliata has been
established from nodal segments taken from 2 years and 10 years old trees but the
successful rooting was achieved only from the nodal segments taken from 2 year
old tree (Mroginski et al., 2003). This loss of rooting ability has also observed in
several other woody species when the explants came from adult plants (Monteuuis,
1987; Capuana and Gianini, 1997). Nodal segments taken from 11-15th position
proved to the best explants for regeneration of Aegle marmelos (Pati et al., 2008).
Cotyledonary node explants of Acacia senegal cultured on induction medium containing
growth regulators gave the highest in number and longest in vitro regenerated shoot
compared to those induced from nodal segment cultured on the same media (Khalafalla and
Daffalla, 2008). The morphogenetic capacity of cotyledonary node explants of
Parapiptadenia rigida was superior to that of nodal segments (Kielse et al., 2009). In
Sapindus trifoliatus, the 4-week-old seedling explants showed maximum shoot proliferation
as compared to 1, 2, and 3 or 5week-old seedling explants (Asthana et al., 2011). The mature
15
nodal explants of Maerua oblongifolia selected from the adult plants showed poor response
as compared to nodal stem segments prepared from fresh shoots sprouts (Rathore and
Shekhawat, 2011). Explant type has been shown to effect multiple shoot induction in a
number of trees including Dalbergia sisso (Pradhan et al., 1998), Albizia lebbeck (Mamun et
al., 2004), Pterocarpus marsupium (Anis et al., 2005) and Albizia odoratissima (Rajeswari
and Paliwal, 2006).
The orientation of explants also plays an important role in regeneration potential.
The horizontal position of the explants has been reported to promote adventitious
shoot formation in many higher plants (Frett and Smagula, 1983; Pierik, 1987).
McClelland and Smith (1990) reported that the horizontal orientation of explants
produced the more shoots per explants in woody species viz. Amefanchier spicata,
Acer rubrun, Border forsythia, Betula nigra. Similar observations on the influence
of explant orientation have also been made for other tree species including Pyrus
communis (Lane, 1979) and Tamarindus (Jaiwal and Gulati, 1991). The horizontal
orientation of nodal explants of Fraxinus angustifolia reported the highest
multiplication rate (Perez-Parron et al., 1994). The orientation of disc in the floral stem
was the most important factor affecting shoot regeneration in Crinum macowanii (Slabbert et
al., (1995). Bhuyan et al., (1997) reported the maximum shoot proliferation in Murraya
koenigii, when the shoot-forming region was in direct contact with the medium surface or
slightly embedded into the medium. Similarly, Bansal and Chibbar (2000) also observed that
vertically placed nodal segments of Madhuca latifolia differentiated more shoots than
explants placed horizontally. Sometimes the vertically placed nodal explants differentiated
more shoots than explants placed horizontally. This could possibly be due to the influence on
polarity of growth regulator transport and directed supply of nutrients (Durzan, 1984).
Shimada et al., (2007) reported that the frequency of adventitious bud formation in Begonia
was dependent on the position of leaf explants on the medium. The abaxial orientation of leaf
explants of Bacopa monnieri on medium can induce fast shoot bud regeneration (Joshi et al.,
2010).
There is great importance of the size of explants to be cultured. Larger the explants,
poor will be the response. The small explants are more easily directed by the substances
contained in the medium. Okazova et al., (1967) reported that small explants are more likely
to form callus while larger explants maintain greater morphogenetic potentiality. This may be
due to the available food reserves and growth regulators which proved useful in the initiation
16
of new growth (Anderson, 1980). Pati et al., (2008) also reported that 3 cm long nodes having
one axillary bud gave quicker bud induction in Aegle marmelos.
Sometimes, the reproductive parts were also used for the regeneration. Regeneration
through inflorescence calli was observed in Aerva tomentosa (Murgai, 1959). Vazquez and
Short, (1978) reported the callus induction from floral parts of African violet. Immature
inflorescences of ginger resulted in the conversion of floral buds into plantlets directly
without intervening callus phase when cultured on MS medium (Babu et al., 1992). Zhong et
al., (1993) developed the regeneration protocol from inflorescence pieces of Beta maritima.
The highest numbers of shoots were regenerated from immature floral stems of Crinum
macowanii (Slabbert et al., 1995). Hannweg et al., (1996) cultured the pieces of inflorescence
stems of Bowiea volubilis. Nirmal and Sehgal (1999) has been developed the protocol for the
micropropagation of Ocimum sanctum through young inflorescence explants. Salvi et al.,
(2000) observed the shoot proliferation from immature inflorescence of Curcum longa. Tyagi
et al., (2005) induced, direct somatic embryogenesis from mature zygotic embryos of
Capparis decidua. Asnita and Norzulaani (2006) reported shoot-like structures from the male
inflorescence of Musa acuminata cultured on MS medium supplemented with BAP.
Shankramurthy and Ksishna (2006) observed the luxuriant mass of callus on MS medium
supplemented with IBA and Kinetin from the immature ovaries of the inflorescence segments
of Embelia ribes. Sharma and Mohan (2006) developed a novel method of shoot regeneration
of Chlorophytum borivilianum from immature floral buds. Inflorescence apices are suitable
explants for the rapid in vitro propagation of Musa species (Resmi and Nair, 2007). Nirmal
and Sehgal (2010) reported the regeneration of Ocimum sanctum using young inflorescences
of mature plants.
2.1.2 Culture media
Growth and morphogenesis of plant tissues in vitro are largely governed by the composition
of the culture media. Although the basic requirements of the cultured plant tissues are similar
to those of whole plants, in practice, nutritional components promoting optimal growth of a
tissue under laboratory conditions may vary with respect to the particular species. Media
compositions are therefore formulated considering specific requirements of a particular
culture system and a number of media have been devised for specific tissues and organs. In
vitro growth of plants is largely determined by the composition of the culture medium. The
importance of nutrition in plant tissue culture is initially reported by Gautheret (1955). The
main components of most plants tissue culture media are mineral salts, sugar as carbon
17
source and water. Other components may include organic supplements, growth regulators and
a gelling agent (Gamborg et al., 1968; Gamborg and Phillips, 1995). Although, the amounts
of the various ingredients in the medium vary for different stages of culture and plant species.
The basic MS (Murashige and Skoog, 1962) and LS (Linsmaier and Skoog, 1965) are most
widely used media. During the past decades, many types of media have been developed in
plant tissue culture (Street and Shillito, 1977; Pierik, 1989; Torres, 1989). Media
compositions have been formulated for the specific plants and tissues (Nitsch and Nitsch,
1969). Some tissues respond much better on solid media while others on liquid media. As
such, no single medium can be suggested as being entirely satisfactory for all types of plant
tissues and organs. Different culture media are proposed by the different scientists from time
to time are varies in salt concentrations from each other’s. Some of the earliest plant tissue
culture media were developed by White (1943) and Gautheret (1939). All subsequent media
formulations are based on White’s and Gautheret’s media. Humidity in the culture vessel and
osmotic potential of the medium affects the growth and development of plantlets in vitro in
different ways (Brown et al., 1979; Ziv et al., 1983).
Some common media used to fulfill the requirements of cultured tissue are MS (Murashigue
and Skoog, 1962), Gamborg (1968), Nitsch and Nitsch (1969), Gressholf and Doy (1972),
Eeuwens (1976), Llyod and McCown (1980), Branton and Blake (1983). Murashique and
Skoog (1962) is the most widely used medium, especially in procedures where plant
regeneration is the main objective.
There are some examples where modified MS medium have also been used viz.
Moringa pterygosperma (Mohan et al., 1995), Hovenia dulcis (Echeverrigaray et
al., 1998), Lagerstroemia reginae (Sumana and Kaveriappa, 2000), Bambusa
vulgaris (Ndiaye et al., 2006). Adventitious shoots were induced from the hypocotyl
explants of Sesbania rostrata on Nitsch’s medium (Nitsch, 1969). Mukhopadhyay and
MohanRam (1981) used Gamborg's B5 medium for the multiplication of Dalbergia
sissoo. Whereas Datta and Datta (1983) obtained multiple shoots from nodal
explants of Dalbergia sissoo on MS medium supplemented with vitamins of
Gamborg's B5 medium and NAA. Reddy et al., (1987) also used MS and B5 media
for callus initiation in Ricinus communis. Muralidharan and Mascarenhas (1987)
reported somatic embryogenesis in Eucalyptus citriodora on semisolid agar based B5 medium
supplemented with NAA and increased sucrose concentration (5%). Dewan et al., (1992) also
observed the higher number of shoots (6.3) on B5 medium in Acacia nilotica. Sarasan et
al., (1994) used MS and B5 medium for induction of callus and somatic
18
embryogenesis in Hemidesmus indicus. Agretious et al., (1996) was observed
better shoot multiplication on MS medium as compared on B5 and White medium
for regeneration in Alpinia calcarata. Babu et al., (2000) cultured nodal explants of
Murraya koengii for regeneration on Woody plant basal medium (WPM). Bhargava et al.,
(2003) reported the formation of globular proembryonic mass of callus on MS
medium after 40-50 days of incubation, and then transfer to B5 medium for fragile
snowy callus in Phoenix dactylifera. Sharada et al., (2003) used MS medium for
shoot induction and B5 or WPM medium for root development in Celastrus
paniculatus.
The superiority of WPM medium on the regeneration was reported in different plants by
different workers viz., Annona squamosa (Lemos and Blake, 1996), Prunus armenica
(Tornero et al., 2000), Quercus floribunda (Purohit et al., 2002) Camellia reticulata (Jose
and Vieitez, 2003), Cinnamomum camphora (Babu et al., 2003) Vitis thunbergii (Lu, 2005),
Garcinia indica (Chabukswar and Deodhar, 2006), Capparis spinosa (Musallam et al., 2011),
Salix tetrasperma (Khan et al., 2011).
The highest frequency of shoot bud proliferation from the cultures of Crataeva
adansonii was observed on MS medium followed by LS medium (Sharma et al.,
2003). Purohit and Kukda (2004) reported the maximum number of shoots on MS
medium followed by SH, WP, B5, and White’s media in Wrightia tinctoria. MS
medium was the most effective for in vitro shoot multiplication from nodal
explants of Holarrhena antidysenterica, amongst the five different basal media assayed
viz. B5, Nitsch, WPM, MS, and Knop’s media (Kumar et al., 2005). WPM medium was
found to be superior to MS medium for the induction of multiple shoots in Tinospora
cordifolia (Raghu et al., 2006). Moreover, shoots of Mucuna pruriens were much longer and
more vigorous and produced more in number on half-strength MS medium than in B5
medium (Faisal et al., 2006). Sotelo and Monza (2007) reported that the shoots of Eucalyptus
maidenii developed on the Quoirin and Lepoivre (1977) basal medium had the best shape,
size and colour. Chorabik (2007) used the two types of media (MS and SH) containing macro
and microelements, enriched with myoinositol glutamine, casein hydrolysate, vitamins and
sucrose for the micropropagation of Abies grandis. Multiple shoots were induced from the
nodal segments of Syzigium cumini inoculated on WPM medium (Remashree et al., 2007).
Tamta et al., (2008) reported the highest numbers of shoots regenerated in Quercus
semecarpifolia on WPM medium. Hazeena and Sulekha (2008) used MS medium for callus
induction and plantlet regeneration using cotyledons explants of Aegle marmelos. Park et al.,
19
(2008) reported adventitious shoot formation in Salix pseudolasiogyne in WPM medium
supplemented with BAP, Zeatin and GA3. Lamrioui et al., (2009) tried MS (1962), Quoirin
and Lepoivre (1977) and Knop (1965) medium for the in vitro germination of Prunus avium.
Thirunavoukkarasu et al., (2010) used MS and WPM medium for plantlet regeneration
through nodal segments of Dalbergia sissoo. MS and WPM medium with different
combinations of cytokinins and auxins were tried for different phases of micropropagation of
Fraxinus micrantha (Bisht et al., 2011). The plantlets of Punica granatum regenerated on MS
medium were found to have better survival compared to WPM medium (Patil et al., 2011).
Bhore and Preveena (2011) used MS, N6 and B5 media to initiate in vitro cultures of
immature zygotic embryos of Mimusops elengi. The best in vitro root development in
Warburgia ugandensis was observed on half strength WPM medium (Kuria et al., 2012).
Plant cells and tissues in culture medium lack autotrophic ability and therefore need external
carbon for energy. The most preferred carbon energy source in plant tissue culture is sucrose.
It is generally used at a concentration of 2-5% while autoclaving the medium sucrose is
converted to glucose and fructose. Sucrose plays an important role in vascular tissue
differentiation. Maltose, galactose, lactose and mannose are the other sources of carbon.
Other carbohydrates may be used occasionally, but none has shown consistent superiority
over sucrose. Ramawat and Arya (1977) studied the effect of carbohydrate on callus culture
of Ephedra gerardiana and Ephedra foliata. Muralidharan and Mascarenhas (1987) reported
somatic embryogenesis in Eucalyptus citriodora on semisolid B5 medium supplemented with
increased sucrose concentration (5%). MS medium supplemented with 2% sucrose was
optimal for culturing of shoot tips of Tamarindus indica (Kopp and Nataraja, 1990). The
supplementation 2-6% sucrose in MS medium supported best root development in Eucalyptus
sideroxylon (Cheng et al., 1992). Marino et al., (1993) reported in vitro proliferation and
rooting capacity of Prunus armeniac on modified MS medium enriched with varying growth
regulator concentrations and sucrose (58.4 mM) or sorbitol (116.8 mM) as main carbon
energy sources. Bennett et al., (1994) reported that 2% sucrose is sufficient for multiplication
and rooting in Eucalyptus globulus. Franca et al., (1995) found that 3% sucrose is effective
for shoot initiation from cotyledonary node of Stryhnodendron polyphythum.
Supplementation of sucrose (3%) and glucose (4%) were the best carbon sources for
proliferation and rooting phases of Quercus suber (Romano et al., 1995). Kumari et al.,
(1998) observed that 2% sucrose has been found more effective for the development of
globular embryos in Terminalia arjuna. Chetia and Handique (2000) found that 3 per cent
sucrose is required for multiple shoot induction in Plumbago indica. Deb (2001) reported the
20
germination of somatic embryos of Melia azedarach on MS medium containing 2 per cent
sucrose. WPM medium supplemented with 1.5% sucrose was optimal for shoot proliferation
from terminal axillary buds of Alnus nepalensis (Thakur et al., 2001). Shahzad and Siddique
(2001) reported that 2-3% sucrose was required for callus induction as well as for shoot
proliferation in Melia azedarach. Among the different carbon sources, i.e., fructose,
galactose, maltose, mannose, and sucrose at 3% (w/v), sucrose supported the best caulogenic
response in Sesbania rostrata (Jha et al., 2002). The medium containing 88 mM of sucrose
was used for the multiplication of Tectona grandis (Gangopadhyay et al., (2003). The highest
number of somatic embryos in Larix leptolepis was noticed on medium containing 0.2M
maltose (Kim and Moon, 2007). Parkash and Staden (2008) reported the induction of
maximum number of shoots from nodal explants of Searsia dentata on MS medium
containing 3% sucrose. The medium containing 4.0% sucrose significantly increased the
number of secondary embryos in Juglans regia (Vahdati et al., 2008). Jain et al., (2008)
reported that, 3 per cent sucrose was preferred carbon source both in terms of growth and
preventing shoot tip necrosis compared to glucose, maltose and fructose at equimolar
concentrations in Harpagophytum procumbens. The supplementation of 2% sucrose was
efficient in plantlet regeneration from the cotyledonary nodes explants of Azadirachta indica
(Reddy et al., 2006), Holarrhena antidysenterica (Mallikarjuna and Rajendrudu, 2009) and
Eucalyptus camaldulensis (Dibax et al., 2010). The best shoot multiplication in Amygdalus
communis was obtained on MS media containing 30 g l-1 sucrose (Akbas et al., 2009).
Plantlets of Simmondsia chinensis regenerated on 0.5% sucrose formed fine and thick roots
(Mills et al., 2009). The MS medium supplemented with higher levels of sucrose (4%)
showed significantly lower frequency of mature somatic embryos in Eucalyptus
camaldulensis, compared to basal medium containing lower concentrations of sucrose
(Prakash and Gurumurthi, 2010). MS medium supplemented with 1.0% sucrose induced the
highest number of somatic embryos in Schisandra chinensis (Chen et al., 2010). Chaari-
Rkhis et al., (2011) used 30 g l-l of mannitol for regeneration in Olea europaea. Gadidasu et
al., (2011) reported the supplementation of 2 % sucrose resulted best organogenesis in
Streblus asper.
Growth additives such as activated charcoal, silver nitrate, silver thiosulphate, ascorbic acid,
jasmonic acid and polyamines cannot strictly be defined as plant growth regulators but they
exert growth modulating effects and may play a novel mean of overcoming recalcitrance
problems of woody plants (Gaspar et al., 1996; Benson, 2000).
21
Addition of yeast extract to MS medium supplemented with NAA and Kn increased
the number of differentiated roots in Dalbergia lanceolaria (Anand and Bir, 1984).
Lakshmi Sita and Shobha Rani (1985) obtained multiple shoots in Eucalyptus grandis on MS
medium supplemented with additional thiamine. Mittal et al., (1989) obtained multiple shoots
from axillary buds of Accacia auriculiformis on Gamborg’s (B5) basal medium supplemented
with coconut milk and BAP. Sita and Swami (1992) also supplemented growth adjuvants like
coconut milk, casein hydrolysate and adenine sulphates to the media for direct organogenesis
and somatic embryogenesis in Dalbergia latifolia. Positive effect of CM in nutrient medium
was developed in Hemidesmus indicus (Sarasan et al., 1994), Elaeocarpus robustus (Roy et
al., 1998). Multiple shoot induction was reported on MS medium supplemented with coconut
milk (5-15%) in Alpinia galanga (Mustafa and Hariharan, 1997) and in Holostemma ada-
kodein (Martin, 2002). Desphande et al., (1998) observed that the MS medium supplemented
with 1-2 mg l-l of adenine sulphate is sufficient for but break and multiple shoot induction in
Ficus riligiosa. Deb (2001) used casein hydrolysate (200 mg l-l) for the induction of
embryogenic callus from imbibed seeds of Melia azedarach. Kaur et al., (1996) reported
maximum shoot bud induction from the cotyledonary nodal explants of Acacia senegal on
MS medium supplemented with adenine sulphate (25.0 mg l-1), ascorbic acid (10.0 mg l-1)
and glutamine (146.0 mg l-1
). Gangopadhyay et al., (2003) used MS medium supplemented
with 0.27 mM adenine sulphate for the multiplication of Tectona grandis. Shrivastava et al.,
(2006) reported shoot differentiation from the cotyledonary nodes and leaves segments of
Cassia senna when cultured on MS medium supplemented with BA, adenine sulphate as well
as complex nitrogenous supplement namely coconut milk (CM). Maximum shoot
proliferation was achieved from nodal explants on MS medium supplemented 135.7 mM
adenine sulfate (Vengadesan et al., 2003). Reddy et al., (2006) obtained the maximum shoot
proliferation from nodal explant of Azadirachta indica inoculated on MS medium
supplemented with 40 mg l-l adenine sulphate, 100 mg l-l glutamine, 10 mg l-l thamine HCl.
Addition of CH or CM induced the development of shoots with profuse callus in Holarrhena
antidysenterica (Mallikarjuna and Rajendrudu, 2009). The addition of casein hydrolysate
significantly increased the number of shoots per explants in Crataeva nurvala (Babbar et al.,
2009), Pongamia pinnata (Belide et al., 2010). Negi et al., (2011) observed enhanced shoot
growth in Cassia auriculata by adding adenine sulphate (25.0 mg l-1), ascorbic acid (20.0 mg
l-1) and L-glutamine (150.0 mg l-1).
Activated charcoal is commonly used in tissue culture media to improve cell
growth and development (Pan and Staden, 1998; Thomas, 2008). The beneficial
22
effects of AC may be attributed to its irreversible adsorption of inhibitory
compounds in the culture medium and substantially reduce the toxic metabolites,
phenolic exudation and exudate accumulation (Fridberg et al., 1978; Thomas,
2008). Rahman et al., (1993) studied the effects of activated charcoal (0.3 per cent w/v) on
shoot elongation in Caesalpinia pulcherrima. Gulati and Jaiwal (1996) used 1% activated
charcoal in medium for root induction in Dalbergia sissoo. Deshpande et al., (1998) also
observed the effects of activated charcoal (0.3 per cent w/v) on shoot elongation in Ficus
religiosa. Mao et al., (2000) also reported the effect of activated charcoal on in vitro
root induction of Litsea cubeba and Sharda et al., (2003) in Celastrus paniculatus.
Similarly, Dibax et al., (2005) found that in addition to suppressing phenolics and
thus browning, adding activated charcoal to the culture medium for regenerating
Eucalyptus enhanced the elongation of shoots and made the leaves dark green and
vigorous. Beneficial effects of activated charcoal were also found on multiple
shoot induction from nodal explants of Wattakaka volubilis (Chakradhar and
Pullaiah, 2006). Agarwal and Kanwar (2007) reported maximum root development
in Morus alba on MS basal medium supplemented with 0.005g l-1 activated
charcoal. Khaled et al., (2009) found that the absorption of inhibitory compounds
form medium or explants by AC resulted in rooting in Ficus anastasia. Addition of
activated charcoal into medium significantly improved the growth of regenerated
shoots of Populus trichocarpa (Kang et al., 2009). A high concentration of activated
charcoal (5% w/v) was optimum for the induction of roots from the regenerated plants of
Commiphora mukul (Kant et al., 2010). In Acacia nilotica, the highest number of shoots
and their elongation was achieved when 200mg l-l activated charcoal was added in
MS medium (Dhabhai and Batra, 2010). Suranthran et al., (2011) reported the best the
growth and development on MS medium supplemented with 2 g l-l AC which significantly
increased plantlet height as well as root length in Elaeis guineensis. The supplementation of
2g l-1
activated charcoal in the culture medium is optimum for shoot elongation in Ocotea
porosa (Pelegrini et al., 2011). Highest root development response of regenerated shoots of
Morus macroura was observed in half strength MS medium supplemented with 4 µM IBA
and 0.1% activated charcoal (Akram and Aftab, 2012).
Gelling and solidifying agents are commonly used for preparing semisolid or solid tissue
culture media. Agar-agar is the most commonly used at the concentration of 0.8% in culture
medium. Use of high concentration of agar makes the medium hard and prevents the
diffusion of nutrients into tissues. In employing a gel medium it is significant to
23
consider both the gel concentration and the quality of the gelling agent. High
concentration of agar, resulting in an excessively hard gel, can inhibit growth of
excised plant tissues. The required concentration of agar should be established
systematically by considering specific needs of each case. Yamuna et al., (1993)
used 6 % agar during regeneration of Cephaelis ipecacuanha. Echeverrigaray et
al., (1998) used MS medium supplemented with 0.7 % agar for the regeneration of
Hovenia dulcis. Naik et al., (2000) used 6% agar-agar during the micropropagation of
Punica granatum. Mroginski et al., (2003) added 0.7% agar to solidify the media used for
micropropagation of Toona ciliata. Uddin et al., (2005) used 5.0 g l-l agar for the
solidification of media used for the micropropagation of Peltophorum pterocarpum. MS
medium solidified with 10 g l-1 of agar was used for the multiplication of Eucalyptus
maidenii (Sotelo and Monza, 2007). All the media were solidified with 0.7%
bacteriological agar-agar for the regeneration of Stereospermum personatum
(Shukla et al., 2009). Supplementation of 6 g l-l agar was resulted best
solidification response and culture establishment in Eucalyptus camaldulensis (Dibax
et al., 2010). Addition of 0.7% agar-agar to the medium resulted best results in Boscia
senegalensis (Khalafalla et al., 2011). Bisht et al., (2011) used MS and WPM medium
containing 0.4% agar for the culture establishment of Fraxinus micrantha.
Plant cells and tissues require optimum pH for growth and development in cultures. The pH
affects the uptake of ions, hence it must be adjusted in between 5-6.0 by adding 0.1N NaOH
(or) Hcl usually the pH higher than six results in a fairly hard medium whereas pH below five
does not allow satisfactory solidification of medium.
2.1.3 Cultural conditions
Light and temperature are the major environmental factors which effects the
vascular tissue differentiation. Most of the cultures grow well within a wide range
of photoperiods, light intensities and optimal temperature (White and Risser, 1964)
whereas some cultures are temperature sensitive (Staritsky, 1970). Cultures are
usually maintained at a constant temperature around 25±20C and 16 hrs
photoperiod followed by 8hrs dark. It appears that the best light exposure period
for a given tissue culture is dependent upon the intensity of the illumination
employed, and probably other factors. Light intensity has influence on biological
effectiveness of the growth regulators added to the growth medium as well as to
affect the endogenous hormone balance in the tissues. When constant conditions are
satisfactory, it may still be necessary to establish optimum temperatures for specific cases.
24
Nitsch and Nitsch (1967) observed that 9-hr daily exposure period using 7000 lux
intensity, regenerated maximum number of shoots in Plumbgo. Calleberg and
Johansson (1993) studied that the direct regeneration was stimulated when the anther cultures
were incubated at 200C. Gupta et al., (1981) obtained multiple shoots from 20 year
old tree of Eucalyptus citriodora when cultured on MS medium incubated at 150C
in continuous light followed by incubating culture at 250C in 16 hours photoperiod.
A high rate of multiplication in Eucalyptus tereticonis has been achieved on MS medium at a
slightly higher temperature (30-320C) (Das and Mitra, 1990). The cultures of Capparis
decidua were incubated at 28 ± 20C with 60% relative humidity and 35-43 µ mol m-2 s-1
photon flux density for 12 h/d photoperiod (Deora and Shekhawat, 1995). All the cultures of
Triphyophyllum peltatum were kept under a 14/10-h (day/night) photoperiod (Bringmann and
Rischer, 2001). Cultures of Melia azedarch were incubated in light at 12-h photoperiod (Deb,
2001). Rajore et al., (2002) reported the multiple shoot formation from nodal
explants of Jatropha curcas inoculated on MS medium incubated at 25±2°C for 16
hours photoperiod. The cultures of Phoenix dactylifera were shifted from dark to
14hr. photoperiod for callus induction (Bhargava et al., 2003). All the cultures of
Andrographis paniculatus were incubated at 22±2 0C provided with 12-h of photoperiod
(Nagaraja et al., 2003). Best growth in cultures of Capparis decidua was reported at 28±10C
(Tyagi et al., 2005). Raghu et al., (2006) noticed best response in Tinospora cordifolia when
incubated under a 10-h photoperiod. Dibax et al., (2010) maintained the cultures of
Eucalyptus camaldulensis in darkness in the growth chamber for 60 days. Girijashankar
(2011) incubated the cultures of Acacia auriculiformis under 16 h photoperiod and 8 h dark
with light intensity of 50µE/m2/s provided by white fluorescent tube lights and at the
temperature 28±2°C. Negi et al., (2011) applied a light regime of 14 hours with 100µmol m-2
s-1
light intensity provided by cool-white fluorescent tubes at 25± 2ºC followed by 10 hrs.
dark period to the cultures of Cassia auriculata. Different temperature conditions ranging
from 23 to 30°C were provided to the cultures of Dalbergia sissoo, however, optimum results
were obtained at 26±1°C and 16/8 h (light/dark) photoperiod (Ali et al., 2012).
2.1.4 Plant growth regulator
Regulation of developmental process in plant tissue culture generally requires the addition of
plant growth regulators to the medium. Plant growth regulators have a significant influence
on shoot regeneration during the initial induction phase (Matt and Jehle, 2005). The growth,
differentiation and organogenesis of tissues become feasible only on the addition of one or
25
more plant regulators to a medium. The ratio of growth regulators required for root and shoot
induction varies considerably with the tissue, which seems directly correlated to the quantum
of growth regulators synthesized at endogenous levels within the cells of the explants
(Razdan, 2003). The age of the mother plant, the conditions under which it has been growing
and the season at which explants are taken, are influenced by the level of naturally occurring
auxins in them (Cassells, 1979). Skoog and Miller (1957) reported that morphogenesis of in
vitro cultured tissues as well as plant development were regulated by plant growth regulators
especially auxins and cytokinins.
Auxins promote cell division and root differentiation. IBA, NAA, IAA, 2, 4-D, etc. are very
widely used as auxins in micropropagation and are incorporated into nutrient media to
promote the growth of the callus, cell suspensions or organs and to regulate morphogenesis,
especially in conjunction with cytokinin. Cytokinins like BAP, Kinetin, Zeatin, etc. are
responsible for all cell division and shoot differentiation. BAP has been the most effective
cytokinin for shoot tip meristem and bud cultures followed by Kinetin (Murashige, 1974).
Cytokinin has been regularly incorporated into tissue culture for shoot regeneration (George
and Sherrington, 1984). The ratio of the auxin to the cytokinin determining the type
of culture established or regenerated, a high auxin to cytokinin ratio generally
favours root formation, whereas a high cytokinin to auxin ratio favours shoot
formation and intermediate ratio favours callus production.
Sehgal (1975) cultured the leaf and petiole segments of Begonia
semperflorens on modified White's basal medium supplemented with various
growth regulators. Goyal and Arya (1979) observed the regeneration in Posopis cineraria
on MS medium supplemented with different concentrations and combinations of Kinetin,
IAA, IBA and BAP. Nodal explants of Prunus cerasus responded best on MS
medium fortified with BAP, IBA and GA3 (Curovic and Ruzi, 1987). Muralidharan
and Mascarenhas (1987) reported somatic embryogenesis in Eucalyptus citriodora on B5
medium supplemented with NAA. Kopp and Nataraj (1990) regenerated plantlets of
Tamarindus indica on MS medium supplemented with 2.0 mg l-l BAP. Multiple
shoots were obtained from cotyledonary nodes of Dalbergia latifolia on MS
medium fortified with BAP (Sita and Swamy, 1992). Reddy et al., (1998) observed the
maximum number of shoots from mature nodal explants of Gymnema sylvestre, on the
medium containing BAP (5.0 mg l-l) and NAA (0.2 mg l-l). Ajithkumar and Seeni (1998)
also reported that in Aegle marmelos, BAP produced longer shoot than Kinetin.
Kathiravan and Ignacimuthu (1990) reported that the combination of BAP and
26
Kinetin produced maximum number of shoots in Canavalia virosa.
Supplementation of BAP (1.5 mg l-l) + NAA (0.1 mg l-l) have been found to show a
good response of shoot proliferation in Vitex negundo (Thiruvengadam and
Jayabalan, 2001). Multiple shoots were initiated from cotyledonary nodes excised from in
vitro grown seedlings of Acacia catechu on MS medium adjuvanted with 1 to 100 µM of BA
(Sahni and Gupta, 2002). Rathore et al., (2004) noticed the multiple shoot formation from the
nodal explants of Syzygium cuminii on MS medium supplemented with BAP (9.0 µM).
Tayagi et al., (2005) reported that the fortification of 2, 4-D induced callus mediated
embryogenesis in Capparis decidua. Gopi and Vatsala (2006) reported the potential of 2, 4-D
(0.1-5.0 mg l-l) + NAA on callus induction in Gymnema sylvestre. Chabukswar and Deodhar
(2006) reported multiple shoot induction in Garcinia indica in WPM medium supplemented
with 8.9 µM BA and 0.5 µM thidiazuron (TDZ). Singh and Lal (2007) reported that media
supplemented with BAP (1.0 mg l-l) in combination with NAA (2.0 mg l-l) supported hundred
per cent callus induction from hypocotyl and cotyledonary leaf segments of Leucaena
leucocephala. Siddique and Anis, (2007) developed an efficient, rapid and
reproducible plant regeneration protocol using nodal explants of Cassia
angustifolia cultured on MS medium supplemented with BAP and TDZ. The
maximum per cent regeneration in Olea europaea was achieved on the medium
supplemented 2.22 µM BAP (Peixe et al., 2007). Kalimuthu et al., (2007) developed a
regeneration protocol for Jatropha curcas using nodal explants on MS supplemented with
BAP (1.5 mg l-1
), Kn (0.5 mg l-1
) and IAA (0.1 mg l-1
). The maximum numbers of shoots in
Aegle marmelos were obtained on the medium supplemented with 8.84 µM BAP in
combination with 5.7 µM IAA (Pati et al., 2008). The best shoot regeneration from the callus
of Aegle marmelos was obtained on MS medium containing 8.8 µM BA and 2.85 µM IAA
(Hazeena and Sulekha, 2008). The highest shoot regeneration frequency as well as shoot
length was induced from nodal explants of Pterocarpus marsupium on MS medium amended
with 4.0 µM BA, 0.5 µM IAA and 20 µM adenine sulphate (Husain et al., 2008). Nodal
explants of Searsia dentata produced multiple shoots when cultured on MS medium
supplemented with 0-2.5 µM BA (Prakash and Staden, 2008). Best shoot induction response
was observed from the nodal explants of Ficus religiosa on MS basal medium supplemented
with 0.5 mg l-1 BAP + 0.1 mg l-1 IAA (Hassan et al., 2009). High shoot multiplication was
observed from axillary buds of Oroxylum indicum inoculated on MS medium fortified with
4.43 µM BAP (Gokhale and Bansal, 2009) and from axillary buds of Morus alba inoculated
on the medium supplemented with 0.5mg l-1 BAP (Balakrishnan et al., 2009). Prakash and
27
Gurumuurthi (2010) obtained callus from zygotic embryos of Eucalyptus camaldulensis
inoculated on MS medium containing 0.5 mg l-l BAP + 0.1 mg l-l NAA. MS medium
containing 3 mg l-1 BA and 0.05-0.1 mg l-1 NAA was most effective in induction of shoots
from nodal explants and shoot tips of Crataeva adansonii (Tyagi et al., 2010). The nodal
explants of Dalbergia sissoo exhibited maximum response on the MS medium fortified with
6.6 µM BA + 1.14 µM IAA (Thirunavoukkarasu et al., 2010). Okere and Adegeye (2011)
reported the best in vitro regeneration in Khaya grandifoilolia on MS medium supplemented
with 1.0 mg l-l BAP + 0.1 mg l-l NAA and 10 mg l-l of adenine sulphate. Zygotic embryos of
Gomortega keule were cultured on WPM medium supplemented with 0.1 mg l-1 NAA and 1.0
mg l-1 BAP (Mun˜oz-Concha and Davey, 2011). Maximum per cent shoot induction and
multiplication in Maerua oblongifolia was achieved on MS medium containing 2.0 mg l-1
BAP (Rathore and Shekhawat, 2011). The media supplemented with 0.2-2 mg l-1 BAP + 0.1-
1 mg l-1 NAA was sufficient for establishment of the culture of Punica granatum (Patil et al.,
2011). Ali et al., (2012) obtained the best in vitro shoot induction in Dalbergia sissoo on MS
medium containing 1.0 mg l-l BAP + 0.25 mg l
-l NAA. The highest numbers of shoots were
resulted from shoot tip explant of Balanites aegyptiaca on medium supplemented with 1.0
mg l-1 Kinetin + 0.2 mg l-1 NAA (El-Mekawy et al., 2012). Amina et al., (2012) observed
multiple shoots from the nodal explants of Helianthemum lippii on MS medium containing
BAP (0.25- 2.0 mg l-1).
2.1.5 Organogenesis
Organogenesis is the de novo production of plant organs (buds, shoots and roots) from
organized tissues or callus. Organogenesis refers to the development of adventitious organs
or primordia from undifferentiated cell mass in tissue culture by the process of
differentiation. In contrast to axillary bud proliferation, organogenesis proceeds de novo via
organization of meristems. It involves the induction of localized meristematic activity by
treatment with plant growth regulators. This leads to the formation of primordium and
eventually the formation of shoot. Micropropagation may be accomplished by either
organogenesis or somatic embryogenesis. Organogenesis involves differentiation of
microshoots and root at different time periods during plantlets development. Usually
microshoots are induced on tissues in a cytokinin rich medium and subsequently microshoots
are rooted in an auxin-enriched medium to give rise to plantlets. Organogenesis has been
introduced on callus, organ, cell and protoplast cultures in plants. However organogenesis is
greatly influenced by the genotype, physiological state of the explants, age of the explants
28
and the in vitro environment, both the light and temperature and the composition of the
medium, in particular plant growth regulators concentration.
Direct Organogenesis
Propagation through axillary bud multiplication is an easy and safe method for obtaining
uniformity and it also assures the consistent production of true-to- type plants within a short
span of time (George, 1993; Salvi et al., 2001). Direct organogenesis is regarded as the most
reliable method for clonal propagation because it upholds genetic uniformity among the
progenies (Beegam et al., 2007).
The direct organogenesis is reported in different tree species by different workers viz.
Eucalyptus torelliana and Eucalyptus camaldulensis (Gupta et al., 1983), Ficus religiosa
(Deshpande et al., 1998), Camellia sp. (Rajkumar and Marimuthu, 2000), Anacardium
occidentale (Hedge et al., 2000), Morus alba (Anis et al., 2003).
In Prosopis cineraria supplementation of Kinetin in combination with IAA induced
higher rate of shoot multiplication than BAP (Goyal and Arya, 1979; 1984). Direct
adventitious shoot formation in Cinnamomum verum was achieved on MS medium
supplemented with 0.1-1.0 mg l-1 Kinetin and BAP (Rai and Chandra, 1987). Mittal et al.,
(1989) observed the multiple shoots induction from cotyledonary nodes of Acacia
auriculiformis. Inomoto and Kitani (1989) developed a micropropagation protocol through
nodal segments of Cinnamomum aromaticum. Dass and Mitra (1990) reported maximum
number of shoots from nodal explants of Eucalyptus tereticornis inoculated on MS medium
supplemented with BAP (1.0 mg l-l) and NAA (0.1 mg l
-l). Mathew and Hariharan (1990)
also reported the adventitious shoot formation from the nodal segments of Syzygium
aromaticum. Rout and Das (1993) reported the multiple shoots induction from apical and
axillary meristems derived from seedlings of Madhuca longifolia. Singh et al., (1993)
observed direct regeneration from axillary bud of Acacia nilotica on MS and WPM medium
fortified with BAP (1.0 mg l-l). In vitro propagation of Moringa pterygosperma from
cotyledonary explants was achieved by Mohan et al., (1995). High-frequency shoot
proliferation was induced from intact seedlings of Murraya koenigii on MS medium
supplemented with 5.0 mg l-l BA (Bhuyan et al., 1997). Sharma and Padhya (1996) also
found the rapid multiplication of Crataeva nurvala through axillary buds on MS medium
supplemented Kinetin and BAP. Direct adventitious shoot formation in Cinnamomum verum
was achieved in WPM medium supplemented with 0.5-4 mg l-1 BAP and Kinetin (Mini et al.,
1997). Reddy et al., (1998) observed the maximum number of shoots from mature nodal
29
explants of Gymnema sylvestre on the medium containing BAP (5.0 mg l-l) and NAA (0.2 mg
l-l). Similarly, Ajithkumar and Seeni (1998) also achieved rapid clonal multiplication of Aegle
marmelos by enhanced axillary bud proliferation on MS medium supplemented with BAP
(2.5 mg l-l) + IAA (1.0 mg l
-l). Jagadishchandra et al., (1999) obtained multiple shoots from
the axillary buds of Pisonia alba on MS medium supplemented with BAP and Kinetin.
Philomina and Rao (2000) reported multiple shoot induction from apical and axillary
meristems of Sapindus mukorossi. Direct adventitious shoot formation in Cinnamomum
verum was achieved on WPM medium supplemented with 0.5-2 mg l-1 BAP and Kinetin
(Sheeja et al., 2000). Komalavalli and Rao (2000) established the protocol for direct
regeneration of Gymnema sylvestre through nodal segments. Mathew et al., (2001) reported
multiple shoot induction through the nodal explants of Garcinia indica and Garcinia gummi-
gutta. Maximum number of shoots were induced from cotyledonary node explants of Acacia
sinuata on MS medium containing 6.66µM BAP+4.65µM Kn (Vengadesan et al., 2002).
Rajore et al., (2002) reported multiple shoots induction from nodal segments of Jatropha
curcas on MS medium fortified with Kn (2.0 mg l-1
) and IBA (1.5 mg l-l). Shu-Hwa et al.,
(2002) reported the multiple shoot formation from seedling and mature explants of
Cinnamomum kanehirae. A high frequency shoot differentiation from nodal explants of
Morus alba was observed on MS medium supplemented with BAP and Kinetin (Anis et al.,
2003). Ramesh et al., (2005) developed an efficient protocol for the micropropagation of
Terminalia bellirica using cotyledonary nodes. Rathore et al., (2005) reported the higher
shoot multiplication through the nodal shoot segments of Maerua oblongifolia
inoculated on MS medium supplemented with various growth regulators. A rapid
multiplication of Nyctanthes arbour-tristis through in vitro axillary shoot
proliferation was reported by Siddique et al., (2006). An efficient, rapid and
reproducible plant regeneration protocol was successfully developed for Cassia
angustifolia using nodal explants on MS medium supplemented with BAP and TDZ
(Siddique and Anis, 2007). Direct shoot formation was achieved in Cinnamomum
camphora on MS medium supplemented with 1.0 mg l-1 BAP and 0.05-2.5 mg l-1TDZ
(Soulange et al., 2007). The highest frequency of shoot regeneration via direct
organogenesis was obtained from petiole explants of Populus ciliata on MS
medium supplemented with 1.5 mg l-l Kn and 0.1 mg l-l IAA (Thakur et al., 2008).
Shukla et al., (2009) developed the in vitro plantlet regeneration protocol from seedling
explants of Stereospermum personatum. Direct regeneration through nodal segments was
observed in Dalbergia sissoo (Thirunavoukkarasu et al., 2010). Multiple shoots were induced
30
from shoot tip explants derived from seedlings of Pterocarpus santalinus (Balaraju et al.,
2011). Explants taken from the seedlings of Sapindus trifoloatus yielded the maximum shoot
regeneration frequency on full-strength MS medium supplemented with 1.0 mg l-1 BAP
(Asthana et al., 2011). Shoot proliferation and elongation was achieved from shoot tips of
Warburgia ugandensis cultured on full strength MS medium containing 1.13 mg l-1 BAP and
0.11 mg l-1 Kn (Kuria et al., 2012).
Indirect Organogenesis
Callus production can be induced from a number of explants like leaf, roots, flower parts and
parts of seed. Explants like tuber, shoot tips, hypocotyl, leaf and stem have been used to
initiate callus with morphogenic potential (Chang and Chang, 1998; Manju and Subramanian,
1999; Kelkar and Krishnamoorthy, 1998).
Lakshmi (1979) raised plantlets of Eucalyptus citriodora from cotyledonary callus.
Shoot buds and root formation was observed from hypocotyl callus of Broussonetia
kasinoki (Ohyama and Oka, 1980). Regeneration was obtained from callus in Leucaena
leucocephala (Venketeswaran and Romano, 1982) and in Albizzia lebbeck (Upadhyay and
Chandra, 1983). Internodal segments of Dalbergia latifolia produced callus on MS media
containing IAA alone or IAA and IBA in combination (Rao, 1986). Vigorous callus
formation was noticed in Cinnamomum verum on MS medium supplemented with 2, 4-D and
BAP (Rai and Chandra, 1987). Adventitious shoot regeneration was reported through
callus culture in Eucalyptus camaldulensis (Murlidharan and Mascarenhas, 1987).
The regeneration of plantlets was achieved through callus derived from shoot tips and shoot
segments of Dalbergia latifolia on MS medium containing NAA and BAP (Rai and Chandra,
1988). Gharyal and Maheshwari (1990) observed callus induction from stem and petiole
explants of Cassia fistula and Cassia siamea and its differentiation into shoot buds.
Nandwani and Ramawat (1991) reported the callus induction and plantlet formation in
Prosopis juliflora. In vitro callus induction and plantlet regeneration from leaf tissue
of Aralia elata has been observed by Jhang et al., (1993). Remeshree et al., (1994)
developed the protocol for callus induction and differentiation of Aristolochia bracteolata.
Chakravarty and Goswami (1999) reported the callus initiation and regeneration of
plantlets from epicotyl explants of Citrus acida on MS medium containing BAP
and 2, 4-D. Calli were induced from different explants of Acacia mangium on MS basal
medium containing 9.05 µM 2, 4-D and 13.95 µM Kinetin (Xie and Hong, 2001). The
maximum shoots were regenerated when the callus derived from the nodal segments of
31
Kigelia pinnata transferred to the medium containing 3.0 mM TDZ and 0.5 mM NAA
(Thomas and Puthur, 2004). Callus formation and shoot differentiation was observed from all
explant of Saussurea obvallata cultured on MS medium containing BA and NAA (Dhar and
Joshi, 2005). Kumari and Shivanna (2005) established a protocol for callus induction
and in vitro regeneration of plantlets from calli derived different explants of
Desmodium oojeinense. Agrawal and Sardar (2006) described high frequency shoot
regeneration through leaflet and cotyledon derived calli of Cassia angustifolia.
High frequency of adventitious shoots were achieved from zygotic embryos derived
callus of Taxus wallichiana on ½ WPM basal medium supplemented with 2.5 mg l-1
BA (Datta et al., 2006).
The highest plantlet regeneration from callus was obtained on (1/4) MS medium
supplemented with 0.2 mg l-l Kn in Irvingia gabonesis (Fajimi et al., 2007).
Soulange et al., (2007) reported callus induction from leaf explants of Cinnamomum
camphora on MS medium containing 1.0 mg l-1 BAP + 0.005-5 mg l-1 TDZ. The highest
numbers of shoots were regenerated from the callus derived from leaf segments of Prunus
serotina on WPM medium supplanted with 9.08 µ M TDZ and 0.54 µM NAA (Liu and Pijut,
2008). Hasan et al., (2008) observed the callus induction and shoots differentiation in Cassia
obtusifolia on the medium having 2.0 mg l-1
2, 4-D + 0.2 mg l-1
Kn. Callus induction and
multiple shoot induction was observed from nodal segments of Sarcostemma brevistigma
cultured on MS medium supplemented with BA or Kinein alone or in combination with NAA
(Thomos and Shanker, 2009). Maximum per cent callus induction from leaf segments of
Citrus jambhiri was reported on MS medium supplemented with 2, 4-D (4.0 mg l -1) and
maximum regeneration through callus was noticed on medium fortified with 0.5 mg l-1 NAA
+ 1.0 mg l-1
BA (Savita et al., 2010). The highest frequency of callus induction was observed
from the nodal segments of Ficus religiosa on MS medium supplemented with 2.26 µM 2, 4-
D (Siwach et al., 2011). Friable callus derived through immature embryos of Zelkova sinica
regenerate shoots when cultured on WPM containing 5.4 µM NAA + 9.0 or 11.2 µM BA (Jin
et al., 2012). The medium supplemented with 0.10 mg l-1 BAP was found to be the best
concentration for shoot differentiation from the callus induced from axillary bud explants of
Michelia champaca (Abdelmageed et al., 2012).
32
Somatic embryogenesis
Somatic embryogenesis is defined as a process in which a bipolar structure, resembling a
zygotic embryo, develops from a non-zygotic cell without vascular connection with the
original tissue. Somatic embryos are used for studying regulation of embryo development,
but also as a tool for large scale vegetative propagation. Somatic embryogenesis is a multi-
step regeneration process starting with formation of proembryogenic masses, followed by
somatic embryo formation, maturation, desiccation and plant regeneration.
Somatic embryogenesis has been first reported by Steward et al., (1958) in
the cultures of Daucus carota. Muralidharan and Mascarenhas, (1987) reported somatic
embryogenesis from embryos of Eucalyptus citriodora cultured on semisolid agar based B5
medium supplemented with NAA and increased sucrose (5%) concentration. The somatic
embryogenesis was obtained from callus derived from immature embryos of Aesculus
hippocastanum cultured on MS medium supplemented with 3.0 mg l-1 2, 4-D, 1.0 mg l-1 Kn,
250 mg l-1 CH and 250 mg l-1 proline (Radojevic, 1988). Shoots were regenerated from
the hypocotyl derived calli of Albizia richardiana on B5 Medium supplemented BAP
(Tomar and Gupta, 1988). Zygotic embryos of Eucalyptus citriodora cultured on
medium containing 3.0 mg l-l NAA formed somatic embryos (Murlidharan and
Mascarenhas, 1987).
Immature zygotic embryos have been found to be a better initial material for somatic
embryo induction in many species, such as Prunus avium (Garin et al., 1997), Phoenix
canariensis (Huong et al.,1999), Larix leptolepis (Kim et al., 1999), Myrtle communis
(Canhoto et al., 1999) and Cryptomeria japonica (Igasaki et al., 2003).
Inamdar et al., (1990) reported somatic embryogenesis through callus derived from
shoot apices of Crataeva nurvala on MS medium containing 2,4-D. Somatic
embryogenesis has been achieved from callus derived from young leaf of Thevetia
peruviana, on MS medium containing 9 µM 2,4-D and 4.6 µM Kn (Kumar, 1992). The calli
transferred to the liquid medium supplemented with 0.5 mg l-1 IAA, formed somatic embryos
after 4 to 5 weeks incubation in Azadirachta Indica (Su et al., 1997). Somatic embryos
were induced from calli derived from hypocotyl explants of Sterculia urens cultured on MS
medium supplemented with 0.45 µM TDZ (Sunnichan et al., 1998). Somatic embryos
were obtained from hypocotyl of mature embryos of Eucalyptus globulus cultured on media
33
containing a high concentration of picloram or IBA, 2, 4-D (Nugent et al., 2001). Somatic
embryogenesis was induced from juvenile explants of Eucalyptus globulus cultured at 24°C
in darkness on MS medium supplemented with different growth regulator combinations
(Pinto et al., 2002). Bhargava et al., (2003) reported regeneration in Phoenix
dactylifera via somatic embryogenesis from callus transferred on MS and B5
medium supplemented with auxins (2, 4-D/IBA/IAA) alone or in combination with
cytokinins. Callus induced from cotyledon and mature zygotic embryo of Terminalia
chebula, produced somatic embryos on MS basal medium supplemented with 50 g l-1 sucrose
(Anjaneyulu et al., 2004). Plant regeneration via somatic embryogenesis was achieved from
embryogenic callus derived from immature zygotic embryos of Azadirachta indica on MS
medium supplemented with 1.11 µM BA and 4.52-6.78 µM 2, 4-D (Rout, 2005). Somatic
embryos were also induced from calli derived from leaflets of Litchi chinensis on
B5 medium containing 4.52 µM 2,4-D and 9.30 µM Kinetin (Raharjo and Litz, 2007).
Kim et al., (2007) obtained somatic embryos directly from cotyledon explants of
zygotic embryos of Podophyllum peltatum cultured on MS medium supplemented
with NAA. Pinto et al., (2008) developed a reproducible protocol for somatic
embryogenesis from mature zygotic embryos of Eucalyptus globulus. The highest induction
frequencies and rapid maturation of somatic embryos was induced from embryogenic calli
derived from the leaf explants of Phœnix dactylifera on MS medium supplemented with 1.0
mg l-1 ABA (Othmani et al., 2009). Somatic embryos were induced from the callus
transferred into MS liquid medium containing NAA (1.0 mg l-l) and BA (1.0 mg l
-l) in
Gymnema sylvestre (Ahmed et al., 2009). Somatic embryos were induced from the calli
derived from petiole and leaf explants of Olea europaea cultured on MS medium without
growth regulators in the dark (Lopes et al., 2009). MS medium supplemented with 12.25 µM
IBA + 4.56 µM Zeatin was the best medium for somatic embryo induction from petiole
derived calli of Olea europaea (Capelo et al., 2010). Shi et al., (2010) reported an efficient
protocol for somatic embryogenesis in Cinnamomum camphora. Somatic embryogenesis in
Gomortega keule was accomplished on MS medium fortified with 1.0 mg l-l 2,4-D and 1.0
mg l-l 2iP (Mun˜oz-Concha et al., 2011). The highest frequency of somatic embryos
induction in Murraya koenigii was recovered from zygotic embryo derived callus transferred
on medium containing 2.27-9.08 µM TDZ (Paul et al., 2011). Somatic embryos were induced
from immature zygotic embryos derived callus of Picea abies cultured on medium
supplemented with 2, 4-D and a cytokinin (Hakman and Arnold, 2012). Somatic embryos
34
were induced from the filaments of Aesculus hippocastanum when cultured on a medium
fortified with 2.5 µm l-l BAP and 5.0 µm 1-1 2, 4-D (Jörgensen, 2012).
2.1.6 Root Development
The development of a perfect plantlet is incomplete without the regeneration of
roots. The occurrence of a phase of dedifferentiation suggests that cells may produce a root
or shoot apical meristem, depending on the added growth regulator during the induction
phase. Root induction was observed in plant growth regulator free Gamborg's B5
medium as well as on 0.1 mg l-1 IAA supplemented medium in Albizzia lebbeck,
Casia fistula and Cassia siamea (Gharyal and Maheshwari, 1990). Regenerated
shoots of Prosopis cineraria developed roots by pulsing with 100 mg l-1
IBA for 4 h
and then culturing on growth regulator free half strength MS medium (Shekhawat et
al., 1993). In Moringa pterygosperma, half strength MS medium supplemented with
GA3 (0.2 mg l-l) produced roots within 7 days in 25 per cent cultures, however,
better rooting was developed with 0.2 mg l-l IBA (Mohan et al., 1995). Sharma and
Padhya (1996) observed root induction from regenerated shoots of Crataeva
nurvala within 7 days on MS medium fortified with 0.5 µ M NAA. Roots were
developed on excised shoots when they were transferred to half-strength MS containing 1.0
mg l-l IBA in Murraya koenigii (Bhuyan et al., 1997). Mustafa and Hariharan (1997)
noticed best root induction from regenerated shoots of Alpinia galanga on MS
medium supplemented with the combinations of NAA and IBA. MS medium (¼)
strength proved most suitable for root induction in Canavalia virosa (Kathiravan
and Ignacimuthu, 1999). A high frequency root development was observed from the
regenerated shoots of Morus indica cultured on medium fortified with 1.0 mg l-1 2, 4-D
(Chitra and Padmaja, 1999). Best rooting in Eucalyptus tereticornis was obtained on half-
strength, MS medium supplemented with 1.0�mg l-1 IBA (Sharma and Ramamurthy, 2000).
Sheeja et al., (2000) reported the root development in Cinnamomum verum on WPM medium
containing 0.5 mg l-1 each IBA and IAA.
Supplementation IBA (2.0 mg l-1) has been found to be effective for root formation
from the excised shoots of Alnus nepalensis (Thakur et al., 2001). In Cardiospermum
halicacabum roots could also be produced in plant growth regulator free media (Babber et al.,
2001). In vitro regenerated shoots of Acacia sinuata induced roots when transferred to half
strength MS medium supplemented with 7.36 µM IBA (Vengadesan et al., 2002). Joshi and
35
Dhar (2003) observed maximum frequency of root development in Saussurea
obvallata on MS half strength medium fortified with 2.5 µ m IBA. Ndoye et al.,
(2003) reported best root development in regenerated shoots of Belanites
aegyptiaca on higher concentration (20 mg l-l) of IBA or NAA. The best root
development was recorded on MS medium supplemented with 1.0 mg l-l NAA in Morus alba
(Anis et al., 2003). Iyer et al., (2005) observed the roots from regenerated shoots of
Michelia champaca on MS medium supplemented with IBA. Regenerated shoots of
Holarrhena antidysenterica were excised and rooted in auxin free MS basal medium
(Mallikarjuna and Rajendrudu, 2007). Vadodaria et al., (2007) found better root formation
on medium containing NAA (0.1 mg l-l) and 1% sucrose in Glycyrrhiza glabra. Higher
percentage of root induction on regenerated shoots of Populus ciliata was obtained on MS
medium supplemented with 0.1 mg l-l IAA (Thakur et al., 2008). Regenerated shoots of
Oroxylum indicum were rooted on half strength MS medium supplemented with 4.92 µM
IBA (Gokhale and Bansal, 2009). Aslam and Khan (2009) observed a high frequency root
development in Phoenix dactylifera on solid MS medium supplemented with 24.6 µM IBA,
however, the root length was higher in liquid medium. The regenerated shoots of Acacia
nilotica were rooted on half strength MS medium fortified with 0.5 mg l-l IBA (Dhabhai and
Batra, 2010). Root induction was observed from regenerated shoots of Aegle marmelos on
MS medium supplemented with 3000 ppm IBA (Warrier et al., 2010). Best rooting response
was observed on half strength MS medium containing IBA (1.0 µM) in Cassia sophera
(Parveen and Shahzad, 2010). Maximum per cent root induction response was obtained by
placing the regenerated shoots of Sapindus trifoliatus in liquid MS medium supplemented
with 1.0 mg l-1 IBA for 24 h and then transferring to the agar solidified MS medium devoid
of IBA (Asthana et al., 2011). Half strength MS basal without supplemented phytohormones
showed best rooting response when compared to all other treatments evaluated in Acacia
auriculiformis (Girijashankar, 2011). The regenerated shoots of Boswellia serrata were
rooted on the medium gelled with 0.6 mg l-l agar (Suthar et al., 2011). The best in vitro root
induction in Warburgia ugandensis was induced on half strength WPM containing 1.0 mg l- l
NAA (Kuria et al., 2012). The rooting response in Pongamia pinnata was enhanced on half-
strength MS media supplemented with 0.5 mg l-1
IBA (Kesari et al., 2012).
2.1.7 Hardening of the regenerants
The heterotrophic mode of nutrition and poor physiological mechanisms and lack of cuticle
on leaves to control water loss, tender the micro propagation plants vulnerable to the
transplantation, plants are acclimatised in suitable compost mixture (or) soil in pots under
36
controlled conditions of light temperature and humidity. Inside the glasshouse the plants
increase their resistance to moisture stress and disease. The plantlets have to become
autotrophic in contrast to their heterotrophic state induced in micropropagation culture.
Transfer of plantlets to soil is the most critical step in micropropagation. The plantlets are
maintained under highly protected conditions in vitro i.e. high humidity, low irradiance, low
CO2 levels and high sugar content.
It is a general observation that the step of transfer from tissue culture vessels to soil is
often very difficult because the in vitro produced plants are not well adapted to an in vivo
climate. Apart from the many adaptation problems of the leaf and shoot systems, the system
of root regeneration in vitro in agar-gelled media appears to be one of the most vulnerable
one. In many cases even negatively gravitropic roots appear in agar gelled media within glass
vessels. Furthermore, the in vitro formed roots do not function properly (fewer root hairs) in
vivo, are rather weak, and often die; in soil, in vitro formed roots often have to be replaced by
newly formed roots. As a consequence of the non-functional roots, transpiration outside the
glass vessels is too high and can result in the loss of many plants. Plants with a good root
system were transferred to small plastic pots containing vermiculite: perlite (1:1)
within a period of 10 days (Deshpande et al., 1996). Daneil et al., (1999) nourishes
the regenerated plantlets of Naregamia alata in vermiculite with a dilute solution
of nutrients. Regenerated plantlets of Litsea cubeba were transplanted into a
potting mixture of sand, loam and peat (1:1:1) for hardening (Mao et al., 2000).
Regenerated plantlets of Eucalyptus tereticornis were hardened in a non-sterile potting mix
at high humidity (Sharma and Ramamurthy, 2000). Sheeja et al., (2000) have reported 43%
establishment of Cinnamomum verum plantlets in soilrite. Rooted shoots of Holostemma
ada-kodien were transferred directly to small pots filled with sterile soilrite and
sand (1:1) ratio (Martin, 2002). In vitro raised rooted plants of Acacia sinuata were
hardened in a growth chamber at 80% relative humidity under 20µmolm-²s-¹ photon lux for
16 hours photoperiod at 35±2˚C (Vengadesan et al., 2003). The acclimatized plantlets of
Syzygium cuminii were transferred to polybags containing mixture of organic manure, garden
soil and sandy soil (1:1:1) for hardening (Rathore et al., 2004). The rooted plantlets of
Terminalia bellereica were transferred to pots and hardened under greenhouse conditions at
65% relative humadity (Ramesh et al., 2005). The regenerated plantlets of Azadirachta indica
were transferred to poly cup containing soil and vermicompost (3:1) and maintained under
high humidity (Reddy et al., 2006). Sharma et al., (2006) used sterilized vermi-compost and
37
soil mixture (1:3) in pots to acclimatize the regenerated plantlets of Vitex negundo .The
regenerated plantlets of Jatropha curcas were hardened on a mixture of decomposed coir
waste, perlite and organic compost in the ratio of 1:1:1 (Kalimuthu et al., 2007). All in vitro
rooted plants of Olea europaea were transferred into jiffy-pots filled with vermiculite-perlite
3:1 (v/v) substrate (Peixe et al., 2007). Soulange et al., (2007) also reported the establishment
of in vitro developed plantlets in pots containing top soil and compost (2:1).
For acclimatization the regenerated plantlets of Sterculia urens were transferred to
plastic cups containing autoclaved vermiculite (Hussain et al., 2008). Rooted shoots
of Aegle marmelos were transferred to glass bottles containing different carrier substrates viz.
autoclaved soil, soil, sand and FYM (1:1:1) and coconut husk and were supplemented with ½
MS plant salt mixture (Pati et al., 2008). Regenerated plantlets of Oroxylum indicum
were initially kept in distilled water in flasks covered with beaker for
approximately 8 days and finally transferred to soil: sand (1:1) in cups (Gokhle and
Bansal, 2009). The regenerated plantlets of Morus alba with well developed roots were
transferred to pots containing soilrite (Balakrishnan et al., 2009). The regenerated shoots
of Taxus baccata were transferred to sterilized soil mixture consisting of peat
moss, sand and soil at the rate of 1:1:1 (Abbasin et al., 2010). Well rooted plantlets
of Commiphora mukul were transferred to glass jars filled with quarter vermiculite
and wetted with Hoagland’s solution (Kant et al., 2010). Regenerated plants of
Gomortega keule were transferred to compost and covered with transparent plastic
bags for acclimatization (Muñoz-Concha and Davey, 2011). Use of coco peat as
hardening medium resulted in maximum survival during hardening phase of Acacia
auriculiformis (Girijashankar, 2011). Regenerated plantlets of Warburgia ugandensis were
watered with half strength WPM on alternate days to harden those (Kuria et al.,
2012).
2.2 Assessment of genetic fidelity using molecular markers
In vitro regeneration of plants involves the application of plant growth regulator, such as
auxins and cytokinins. Nevertheless, these plant growth regulators are known to be associated
with genetic instability in plants (Karp, 1989; Cullis, 1992). Changes to these growth
regulator habituations are known to be associated with genetic instability in plants. It is
believed that high concentrations of plant growth regulators can modify the frequency of
ploidy changes and point mutations. Factors such as explants source, time of culture, number
38
of subcultures, plant growth regulator, genotype, media composition, the level of ploidy are
capable of inducing in vitro variability (Silvarolla, 2000; Yu et al., 2008). Several
mechanisms governing somaclonal variation induced during subculturing includes gene
amplification, single nucleotide base change, transposon migration, altered methylation
states, chromosome instability, chromosome inversion, single gene mutations, translocations,
cytoplasmic genetic changes, ploidy changes, rearrangements and partial chromosome
deletions (Duncan et al., 1986, Yu and Buckler, 2006).
Variation also occurs as responses to the stress imposed on the plant in culture conditions and
are manifested in the form of DNA methylations, chromosome rearrangements and point
mutations (Phillips et al., 1994). Traditionally, morphological description, physiological
supervision, karyological analysis, biochemical estimations and field assessment were used to
detect any types of genetic variations, but presently molecular markers have complemented
over traditional methods to detect and monitor the genetic fidelity of tissue culture derived
plantlets and variety identification. Recently, molecular markers have been used in the
detection of variation or confirmation of genetic fidelity during micropropagation (Gupta et
al., 1998; Tyagi et al., 2007). Molecular markers have been used successfully to determine
the degree of relatedness among individuals or group of accessions to clarify the genetic
structure or variation among accessions, population, varieties and species.
Molecular markers are now routinely used for characterization of genetic diversity, DNA
fingerprinting, genome mapping, genome evolution, ecology, taxonomy, and plant breeding.
DNA-based markers are abundant, highly polymorphic and independent of environment or
tissue type. Most DNA-based markers can be classified into three categories depending on
the technique used (Karp and Edwards, 1997): Hybridization-based DNA markers, arbitrarily
primed polymerase chain reaction (PCR)-based markers, sequence targeted and single locus
DNA markers. Restriction fragment length polymorphism (RFLP) is an hybridization-based
markers in which DNA polymorphism is detected by digesting DNA with restriction enzymes
followed by DNA blotting and hybridizations with probes. Arbitrarily primed PCR-based
markers are employed in organisms for which no genome sequence is available. These
markers are RAPD and AFLP. Sequence tagged sites (STS), SSR, Single nucleotide
polymorphisms (SNP) markers belong to sequence targeted and single locus PCR-based
DNA markers. Among the polymerase chain reaction (PCR) based markers frequently used,
RAPD (random amplified polymorphic DNA) is considered to be efficient and cost effective.
The technique only needs a few nanograms of DNA for a fast polymorphism analysis, does
not require prior knowledge of DNA sequence, and does not involve radioactivity (Williams
39
et al., 1990). Isozyme markers provide a convenient method for detecting genetic changes,
but are subject to ontogenic variations. They are also limited in number, and only DNA
regions coding for soluble proteins can be sampled.
A large number of RFLPs were recorded in some tree species like Populus, eucalyptus etc.,
for studying the variation and genetic fidelity of micropropagated plants. There are a number
of reports in literature which demonstrated the detection of genetic fidelity using RAPD/ISSR
markers in various plant species such as Picea mariana (Isabel, 1993), Festuca pratensis
(Valles et al., 1993), Picea abeies (Heinze and Schmidt, 1995), Popolus deltoides (Rani et
al., 1995), Pinus thunburghii (Goto et al., (1998), Eucalypyus ( De Laia et al., 2000),
Actinidia deliciosa (Palombi and Damiano, 2002), Vitex negundo (Ahmad et al,. 2008)
Dendrocalamus hamiltonii (Agnihotri et al., 2009), Capparis decidua (Tyagi et al., 2010).
Bouman et al., (1992) and Bouman and Kuijpers (1994) also found RAPD polymorphism
amongst micropropagated plants of Begonia. Similarly, Rani et al., (1995) reported that the
plants originating from the same clone of Populus deltodeis showed all the amplification
products were monomorphic across all the micropropagated plants. Absence of genetic
variation using the RAPD marker system has been reported in several cases such as
micropropagated shoots of Pinus thunbergii (Goto et al., 1998), somatic embryogenesis-
derived regenerants of oil palm (Rival et al., 1998), micropropagated teak somatic
embryogenesis-derived regenerants (Gangopadhyay et al., 2003). Similarly, RAPD markers
have been applied for characterization of micropropagated Populus tremuloides (Rahman and
Rajora, 2001). About 97% homology between the mother plants and micropropagated plants
has been reported in Syzygium travancorium (Anand, 2003). Random amplified polymorphic
DNA analysis was resulted 30% polymorphism in Robinia pseudoacacia (Bindia and
Kanwar, 2003). Scocchi et al., (2004) also used the RAPD markers to test the genetic
stability of in vitro raised plants of Melia azedarach. Out of six RAPD and four ISSR primer
combinations used for PCR amplification of in vitro raised plants of Azadirachta indica, a
total of 60 bands were scored of which 48.3% were polymorphic (Kota et al., 2006).
Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic
stability of 80 micropropagated Hagenia abyssinica plants (Feyissa et al. 2007). Similarly, a
total number of 925 bands were obtained from the PCR profile of micropropagated shoots of
Mucuna pruriens (Sathyanarayana et al., 2008). RAPD analysis in 90 micropropagated plants
of Eucalyptus globulus was resulted with a total of 115 amplified reproducible bands per
plant produced from 14 random primers (Liu et al., 2009). Reproducible monomorphic
RAPD banding patterns have been obtained using all the tested primers in Phoenix
40
dactylifera (Othmani et al., 2010). RAPD analysis confirmed that all the in vitro derived
plants of Populus alba and Populus tremula were genetically identical to their donor plants
(Khattab, 2011). Nadha et al., (2011) utilized RAPD and ISSR markers to assure the genetic
fidelity of in vitro raised Guadua angustifolia clones. Analysis of RAPD banding patterns
generated by PCR amplification using 20 random primers gave no evidences for somaclonal
variation in Saccharum officinarum (Pandey et al., 2012).
ISSR technique has successfully been used for the assessment of genetic fidelity in Populus
trimuloides (Rahman and Rajora, 2001), Robina ambigua (Guo et al., 2006b). However, Guo
et al., (2006a) reported 15.7% of polymorphic bands in the ISSR analysis for the 63
regenerants of Codonopsis lanceolata. Amplification of the ISSR markers reported
polymorphism in Actinidia deliciosa (Palombi and Damiano, 2002). By using ISSR, a low
genetic variation (3.92%) among the 21 in vitro grown plants of Dictyospermum ovalifolium
was reported by Chandrika et al., (2008). All ISSR profiles of micropropagated plants were
monomorphic, and similar to those of field-grown plants of Vitex nigundo (Ahmad et al.,
2008), Ochreinauclea missionis (Chandrika and Rai, 2009), Crataeva magna (Bopana and
Saxena, 2009) and Nothapodytes foetida (Chandrika et al., 2010), Phoenix dactylifera
(Kumar et al., 2010), Gentiana straminea (He et al., 2011). Genetic stability of the
regenerated plants of Anisodus tanguticus was assessed by 25 ISSR markers (He et al., 2011).
Liu and Yang (2012) reported that out of 21 ISSR primers screened, 16 primers were found
to produce clear, reproducible bands with an average of 6.5 bands per primer in Psidium
guajava. ISSR banding pattern analysis generated 15 primers (112 amplicons) and gave no
evidences for somaclonal variation in Saccharum officinarum (Pandey et al., 2012).
Vendrame et al., (1999) evaluated the applicability of AFLP analysis for the assessment of
somatic embryos of Carya illinoinensis. Singh et al., (2002) observed that the two hundred
and thirty-nine amplified AFLP fragments were monomorphic across the mother tree and its
tissue culture raised progenies of Azadarachta indica. Devarumath (2002) performed the
RFLP fingerprinting of Camellia chinensis using six restriction endonuclease. Amplified
fragment length polymorphism (AFLP) markers were employed to detect genetic fidelity
between in vitro raised plantlets of Papaver bracteatum and mature (Carolan et al., 2002).
Bhatia et al., (2005) observed that the DNA samples obtained from the regenerated shoots
and cotyledonary explants were subjected to amplified fragment length polymorphism
(AFLP) analysis to examine genetic uniformity of tissue-cultured tomato. Tripathi et al.,
(2006) reported the identification of Eucalyptus clones regenerated through tissue culture by
using genetic markers viz., RAPDs/AFLPs. Analysis of the AFLP banding patterns exhibited
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no variation about the number and the size of AFLP bands either among the somatic embryos
derived plants and the mother plant of Phoenix dactylifera (Othmani et al., 2010). Keivani et
al., (2010) observed polymorphic bands in regenerated plants of Medicago sativa.
In isozyme analyses, the particular enzyme is extracted from the plant tissue, and the different
forms separated by gel electrophoresis, on the basis of molecular size, shape and electrical
charge. Gangopadhyay et al., (2004) observed identical isozymic profiles for mother plant
and tissue cultured plants of Pandanus amaryllifolius using acid phosphatase isozymes.
Garcia et al., (2004) reported the genetic fidelity testing using isozyme markers like as,
shikimate dehydrogenase (SDH), isocitric dehydrogenase (IDH), acid phosphatase (ACP),
malate dehydrogenase (MDH) and glutamate oxaloacetate transaminase (GOT). Scocchi et
al., (2004) also used the isozyme markers to access the genetic stability of in vitro raised
plants of Melia azedarach. Similar reports have been observed in tissue culture derived
plantlets of Celastrus paniculatus and date palm (Maruthi et al., 2004; Saker et al., 2000).
Picoli et al., (2008) has also observed the isozyme patterns of different isozyme like esterase,
peroxidase, acid phosphatase, malate dehydrogenase to access the genetic fidelity of
Eucalyptus. Cheniany et al., (2010) used peroxidase and polyphenol oxidase isozymes as
markers for studying the physiological processes of rooting in Persian walnut. The isozymic
profile indicated the genetic conformity of Naringi crenulata and Aegle marmelos among
plantlets obtained through in vitro propagation and mother plant were all true to the type
(Smila et al., 2011).